Bioactive Platinum(IV) Complexes Incorporating Halogenated Phenylacetates

A new series of cytotoxic platinum(IV) complexes (1–8) incorporating halogenated phenylacetic acid derivatives (4-chlorophenylacetic acid, 4-fluorophenylacetic acid, 4-bromophenylacetic acid and 4-iodophenylacetic acid) were synthesised and characterised using spectroscopic and spectrometric techniques. Complexes 1–8 were assessed on a panel of cell lines including HT29 colon, U87 glioblastoma, MCF-7 breast, A2780 ovarian, H460 lung, A431 skin, Du145 prostate, BE2-C neuroblastoma, SJ-G2 glioblastoma, MIA pancreas, the ADDP-resistant ovarian variant, and the non-tumour-derived MCF10A breast line. The in vitro cytotoxicity results confirmed the superior biological activity of the studied complexes, especially those containing 4-fluorophenylacetic acid and 4-bromophenylacetic acid ligands, namely 4 and 6, eliciting an average GI50 value of 20 nM over the range of cell lines tested. In the Du145 prostate cell line, 4 exhibited the highest degree of potency amongst the derivatives, displaying a GI50 value of 0.7 nM, which makes it 1700-fold more potent than cisplatin (1200 nM) and nearly 7-fold more potent than our lead complex, 56MESS (4.6 nM) in this cell line. Notably, in the ADDP-resistant ovarian variant cell line, 4 (6 nM) was found to be almost 4700-fold more potent than cisplatin. Reduction reaction experiments were also undertaken, along with studies aimed at determining the complexes’ solubility, stability, lipophilicity, and reactive oxygen species production.

Molecules 2022, 27, x FOR PEER REVIEW 2 of 38 multiple cancer types [8][9][10], platinum(II) drugs have undesirable attributes that significantly impact the quality of life for cancer patients [11][12][13][14]. Deviating from the SAR design that created cisplatin derivatives, our research group has previously reported a selection of structurally distinct platinum(II) complexes of the type [Pt II (HL)(AL)] 2+ , where HL is the heterocyclic ligand (i.e., 1,10-phenanthroline (phen) or 5,6-dimethyl-1,10-phenanthroline (5,6-Me2phen)) and AL is a chiral ancillary ligand (1S,2S-diaminocyclohexane (SS-DACH or DACH)) ( Figure 2) [15][16][17][18][19][20]. Amongst all complexes investigated within the class, [Pt II (5,6-Me2phen)(SS-DACH)] 2+ (56MESS) (Figure 2) proved to be the most biologically active, demonstrating superior anticancer killing properties in multiple human cancer cell lines compared to platinum(II) drugs [15][16][17][18]20,21]. Its analogue, [Pt II (phen)(SS-DACH)] 2+ (PHENSS) (Figure 2), also showed significant potency in other human cancer cell lines compared to cisplatin and its derivatives [16,17,[19][20][21]. Interestingly, the mechanism of action of our lead complex, 56MESS, differs from the covalent binding mechanism of clinically used platinum(II) drugs [22]. 56MESS interferes with the mitochondria, cytoskeletal proteins, and to a lesser extent nuclear DNA [23]. Despite evidence that these unconventional platinum(II) complexes exhibit potent cytotoxicity in vitro, there has been mixed evidence that this activity is translated into in vivo studies. For example, BD-IX rats with peritoneal carcinomatosis were treated with 56MESS via intravenous and intraperitoneal methods; this treatment did not elicit a tumour suppression response and at pharmacological doses induced nephrotoxicity [24]. By contrast, in a separate study, mice bearing PC3 (prostate carcinoma) tumour xenografts treated with PHENSS or cisplatin both demonstrated a comparable decrease in mean tumour weight in relation to the control group [19]. Importantly, with no obvious signs of toxicity observed in mice treated with PHENSS, half of the cisplatin-treated mice perished by day 20. Because of this discrepancy between in vitro and in vivo antitumor effects of these unconventional platinum(II) complexes, their corresponding platinum(IV) Despite evidence that these unconventional platinum(II) complexes exhibit potent cytotoxicity in vitro, there has been mixed evidence that this activity is translated into in vivo studies. For example, BD-IX rats with peritoneal carcinomatosis were treated with 56MESS via intravenous and intraperitoneal methods; this treatment did not elicit a tumour suppression response and at pharmacological doses induced nephrotoxicity [24]. By contrast, in a separate study, mice bearing PC3 (prostate carcinoma) tumour xenografts treated with PHENSS or cisplatin both demonstrated a comparable decrease in mean tumour weight in relation to the control group [19]. Importantly, with no obvious signs of toxicity observed in mice treated with PHENSS, half of the cisplatin-treated mice perished by day 20. Because of this discrepancy between in vitro and in vivo antitumor effects of these unconventional platinum(II) complexes, their corresponding platinum(IV) complexes have been explored as a strategy to harness their potential [21,[25][26][27][28][29][30]. Preliminary in vivo data on murine LLC models, administered orally, demonstrated that unconventional platinum(IV) complexes outperformed cisplatin [30].
What makes platinum(IV) complexes attractive as a drug design strategy is their octahedral geometry that allows the inclusion of two additional ligands through coordination of the axial positions [31]. The additional coordination positions offer the ability to design combinatorial therapies in a single complex, which is potentially more effective than the precursor platinum(II) complex. Specifically, platinum(IV) complexes are proposed to be stable in the bloodstream due to their low spin d 6 electronic configuration [32]. It is only once platinum(IV) complexes cross into cancer cells that the platinum(IV) undergoes reduction to the biologically active species with the release of the axial ligands [33], and this makes them effective prodrugs. Normally, reduction occurs in the presence of biological reductants such as ascorbic acid (AsA) or glutathione (GSH) [33,34]. This unique advantage allows platinum(IV) complexes to potentially avoid off-target biomolecules in the body, resulting in fewer side effects and increasing drug selectivity [35]. The effectiveness of this characteristic has been explored with bis-(acetate)-ammine dichloro-(cyclohexylamine) platinum(IV) (satraplatin), cis,trans,cis-dichlorodihydroxo-bis-(isopropylamine) platinum(IV) (iproplatin), and tetrachloro(1R,2R-diaminocyclohexane) platinum(IV) (tetraplatin) (Figure 3). Despite the potential of this approach, no platinum(IV) complexes of this type have been approved for clinical use due to either lack of activity or failure to demonstrate an overall improvement in survival [36].
Molecules 2022, 27, x FOR PEER REVIEW 3 of 38 complexes have been explored as a strategy to harness their potential [21,[25][26][27][28][29][30]. Preliminary in vivo data on murine LLC models, administered orally, demonstrated that unconventional platinum(IV) complexes outperformed cisplatin [30]. What makes platinum(IV) complexes attractive as a drug design strategy is their octahedral geometry that allows the inclusion of two additional ligands through coordination of the axial positions [31]. The additional coordination positions offer the ability to design combinatorial therapies in a single complex, which is potentially more effective than the precursor platinum(II) complex. Specifically, platinum(IV) complexes are proposed to be stable in the bloodstream due to their low spin d 6 electronic configuration [32]. It is only once platinum(IV) complexes cross into cancer cells that the platinum(IV) undergoes reduction to the biologically active species with the release of the axial ligands [33], and this makes them effective prodrugs. Normally, reduction occurs in the presence of biological reductants such as ascorbic acid (AsA) or glutathione (GSH) [33,34]. This unique advantage allows platinum(IV) complexes to potentially avoid off-target biomolecules in the body, resulting in fewer side effects and increasing drug selectivity [35]. The effectiveness of this characteristic has been explored with bis-(acetate)-ammine dichloro-(cyclohexylamine) platinum(IV) (satraplatin), cis,trans,cis-dichlorodihydroxo-bis-(isopropylamine) platinum(IV) (iproplatin), and tetrachloro(1R,2R-diaminocyclohexane) platinum(IV) (tetraplatin) (Figure 3). Despite the potential of this approach, no platinum(IV) complexes of this type have been approved for clinical use due to either lack of activity or failure to demonstrate an overall improvement in survival [36]. Recent studies have focused on developing platinum(IV) complexes in combination with a diverse class of bioactive inhibitors that perturb cancer development, and interestingly, cisplatin remains a popular scaffold [37][38][39][40][41][42]. Histone deacetylase inhibitors, such as 4-phenylbutyric acid and valproic acid (Figure 4), are a class of anticancer agents, which have been coordinated with cisplatin because of their ability to expose DNA to platination [43]. Likewise, non-steroidal anti-inflammatory drugs, such as indomethacin, aspirin, ibuprofen, and naproxen ( Figure 4), have also been conjugated with cisplatin to produce prodrugs that primarily target tumour-associated inflammation [44]. Additionally, dichloroacetic acid (DCA) (Figure 4) is a pyruvate dehydrogenase kinase inhibitor that has been extensively reported in the literature because of its effectiveness in suppressing the Warburg effect (a cancer hallmark) and its synergism with cisplatin [45][46][47][48][49][50]. The success of DCA as co-treatment with other chemotherapeutics has also been widely reported in the literature, despite it not being FDA-approved [46,[50][51][52][53][54]. Furthermore, the DNA alkylating agent, chlorambucil (Figure 4), has also been coordinated with cisplatin and oxaliplatin to generate dual-DNA targeting prodrugs [55][56][57][58]. Recent studies have focused on developing platinum(IV) complexes in combination with a diverse class of bioactive inhibitors that perturb cancer development, and interestingly, cisplatin remains a popular scaffold [37][38][39][40][41][42]. Histone deacetylase inhibitors, such as 4-phenylbutyric acid and valproic acid (Figure 4), are a class of anticancer agents, which have been coordinated with cisplatin because of their ability to expose DNA to platination [43]. Likewise, non-steroidal anti-inflammatory drugs, such as indomethacin, aspirin, ibuprofen, and naproxen ( Figure 4), have also been conjugated with cisplatin to produce prodrugs that primarily target tumour-associated inflammation [44]. Additionally, dichloroacetic acid (DCA) (Figure 4) is a pyruvate dehydrogenase kinase inhibitor that has been extensively reported in the literature because of its effectiveness in suppressing the Warburg effect (a cancer hallmark) and its synergism with cisplatin [45][46][47][48][49][50]. The success of DCA as co-treatment with other chemotherapeutics has also been widely reported in the literature, despite it not being FDA-approved [46,[50][51][52][53][54]. Furthermore, the DNA alkylating agent, chlorambucil (Figure 4), has also been coordinated with cisplatin and oxaliplatin to generate dual-DNA targeting prodrugs [55][56][57][58]. With the many bioactive axial ligands that have been studied with platinum, a naturally occurring auxin substance called phenylacetic acid (PAA) caught our interest ( Figure  5). Apart from being a fundamental block of multiple pharmaceutical intermediates, PAA is also a promising non-toxic drug for cancer treatment [59][60][61][62]. It was suggested that by preserving the aromatic ring and carboxylic group of PAA while adding other structural functionality, more potent derivatives might be generated [59]. In light of this, we introduced a series of halogenated PAA derivatives, including 4-chlorophenylacetic acid (4-CPA), 4-fluorophenylacetic acid (4-FPA), 4-bromophenylacetic acid (4-BPA), and 4-iodophenylacetic acid (4-IPA) ( Figure 5), as axial ligands to the cores of our reactive platinum(IV) scaffold, [Pt IV (HL)(AL)(OH)2] 2+ . This design approach was intended to create multimodal prodrugs and to investigate if the halogens on the axially coordinated phenylacetic acids (chlorine (Cl), fluorine (F), bromine (Br) and iodine (I)) would impact the solubility, stability, reduction property, lipophilicity, overall cytotoxicity, and reactive oxygen species (ROS) potential of the prodrugs.
Amongst the selected axial ligands, 4-CPA was of primary interest because previous studies highlighted its effectiveness against estrogen-induced mammary tumorigenesis [63][64][65]. Estrogen and its receptor are critical factors in both normal breast development and the progress of breast carcinomas [66]. Further evidence suggests that estrogen production in breast tissues is mainly due to the overexpression of aromatase enzymes, especially in the case of postmenopausal breast cancer [67,68]. Aromatase is crucial in estrogen biosynthesis, as it catalyses the aromatization of androgen to form estrogen. Increased aromatase enzyme activity is also found in tumour-expressing breast cancer cells, suggesting such enzymes are viable therapeutic targets [66,68]. Treating estrogen-sensitive breast cancer with current therapies is challenging, as resistance develops after prolonged administration and can involve complications [69]. From this, Sidell et al. studied the efficacy of 4-CPA as a potent aromatase inhibitor and demonstrated a novel mechanism of action by which it antagonizes estrogen signalling [63]. Interestingly, this differs from the mechanism of action of classical anti-breast cancer drugs such as tamoxifen. A prototype platinum(IV) prodrug bearing 4-CPA is expected to express the attributes of both moieties With the many bioactive axial ligands that have been studied with platinum, a naturally occurring auxin substance called phenylacetic acid (PAA) caught our interest ( Figure 5). Apart from being a fundamental block of multiple pharmaceutical intermediates, PAA is also a promising non-toxic drug for cancer treatment [59][60][61][62]. It was suggested that by preserving the aromatic ring and carboxylic group of PAA while adding other structural functionality, more potent derivatives might be generated [59]. In light of this, we introduced a series of halogenated PAA derivatives, including 4-chlorophenylacetic acid (4-CPA), 4-fluorophenylacetic acid (4-FPA), 4-bromophenylacetic acid (4-BPA), and 4-iodophenylacetic acid (4-IPA) ( Figure 5), as axial ligands to the cores of our reactive platinum(IV) scaffold, [Pt IV (H L )(A L )(OH) 2 ] 2+ . This design approach was intended to create multimodal prodrugs and to investigate if the halogens on the axially coordinated phenylacetic acids (chlorine (Cl), fluorine (F), bromine (Br) and iodine (I)) would impact the solubility, stability, reduction property, lipophilicity, overall cytotoxicity, and reactive oxygen species (ROS) potential of the prodrugs.
Amongst the selected axial ligands, 4-CPA was of primary interest because previous studies highlighted its effectiveness against estrogen-induced mammary tumorigenesis [63][64][65]. Estrogen and its receptor are critical factors in both normal breast development and the progress of breast carcinomas [66]. Further evidence suggests that estrogen production in breast tissues is mainly due to the overexpression of aromatase enzymes, especially in the case of postmenopausal breast cancer [67,68]. Aromatase is crucial in estrogen biosynthesis, as it catalyses the aromatization of androgen to form estrogen. Increased aromatase enzyme activity is also found in tumour-expressing breast cancer cells, suggesting such enzymes are viable therapeutic targets [66,68]. Treating estrogen-sensitive breast cancer with current therapies is challenging, as resistance develops after prolonged administration and can involve complications [69]. From this, Sidell et al. studied the efficacy of 4-CPA as a potent aromatase inhibitor and demonstrated a novel mechanism of action by which it antagonizes estrogen signalling [63]. Interestingly, this differs from the mechanism of action of classical anti-breast cancer drugs such as tamoxifen. A prototype platinum(IV) prodrug bearing 4-CPA is expected to express the attributes of both moieties and provide evidence for the effectiveness of this strategy for treating estrogen-sensitive breast cancer.

Synthesis and Characterisaion
All NHS esters were synthesised as previously described with minor modifications [70,71]. Minor modifications refer to solvent choice, reaction time, and modifying extraction or isolation steps to obtain the products with higher yields. Each starting acid was reacted with a dehydrating agent, DCC in C3H6O or DCM to afford a cloudy solution that is primarily due to the presence of an unwanted by-product, dicyclohexylurea (DCU) (Figure 7). The solutions were filtered either by gravity filtration or syringe filtration, collected, and reduced to dryness to afford a crystalline product. Syringe filtration was more efficient than gravity filtration. In some instances, the resulting products were isolated as oils. The products were used without purification in the following reactions. An average yield of ~80% was recorded for all of the NHS esters. Characterisation to confirm the successful synthesis of the esters included HPLC ( Figures   The precursor platinum(II) (PHENSS and 56MESS) and platinum(IV) (PHENSS(IV)(OH)2 and 56MESS(IV)(OH)2) complexes were synthesised as previously described [21,71]. The synthesis of the desired products, 1-8, was initially undertaken

Synthesis and Characterisaion
All NHS esters were synthesised as previously described with minor modifications [70,71]. Minor modifications refer to solvent choice, reaction time, and modifying extraction or isolation steps to obtain the products with higher yields. Each starting acid was reacted with a dehydrating agent, DCC in C 3 H 6 O or DCM to afford a cloudy solution that is primarily due to the presence of an unwanted by-product, dicyclohexylurea (DCU) (Figure 7). The solutions were filtered either by gravity filtration or syringe filtration, collected, and reduced to dryness to afford a crystalline product. Syringe filtration was more efficient than gravity filtration. In some instances, the resulting products were isolated as oils. The products were used without purification in the following reactions. An average yield of 80% was recorded for all of the NHS esters. Characterisation to confirm the successful synthesis of the esters included HPLC ( Figures

Synthesis and Characterisaion
All NHS esters were synthesised as previously described with minor modifications [70,71]. Minor modifications refer to solvent choice, reaction time, and modifying extraction or isolation steps to obtain the products with higher yields. Each starting acid was reacted with a dehydrating agent, DCC in C3H6O or DCM to afford a cloudy solution that is primarily due to the presence of an unwanted by-product, dicyclohexylurea (DCU) (Figure 7). The solutions were filtered either by gravity filtration or syringe filtration, collected, and reduced to dryness to afford a crystalline product. Syringe filtration was more efficient than gravity filtration. In some instances, the resulting products were isolated as oils. The products were used without purification in the following reactions. An average yield of ~80% was recorded for all of the NHS esters. Characterisation to confirm the successful synthesis of the esters included HPLC ( Figures   The precursor platinum(II) (PHENSS and 56MESS) and platinum(IV) (PHENSS(IV)(OH)2 and 56MESS(IV)(OH)2) complexes were synthesised as previously described [21,71]. The synthesis of the desired products, 1-8, was initially undertaken The precursor platinum(II) (PHENSS and 56MESS) and platinum(IV) (PHENSS(IV) (OH) 2 and 56MESS(IV)(OH) 2 ) complexes were synthesised as previously described [21,71]. The synthesis of the desired products, 1-8, was initially undertaken using the methods reported in the literature [30,72,73]. Unfortunately, these methods did not produce the intended complexes reliably (i.e., poor yield, unwanted by-products, and product isolation difficulty); therefore, method development was required to obtain the most efficient method for isolating the complexes [71]. To prepare 1-8, the appropriate precursor, [Pt IV (H L )(A L )(OH) 2 ] 2+ , was reacted with 2-3.5 mol eq. of the NHS esters in DMSO and left stirring for 72 h at room temperature in the dark ( Figure 8). Each reaction was washed with excess Et 2 O to afford two layers: a colourless and an oily brown layer. The colourless layer was discarded while the oily brown layer was collected and diluted with a minimum amount of MeOH, followed by the addition of excess Et 2 O to afford a beige precipitate. The precipitate was collected and washed with excess C 3 H 6 O to remove any unreacted or excess NHS esters and acids. Further purification using flash chromatography was required to obtain higher purity. Characterisation to confirm the successful synthesis  Table 1.
Molecules 2022, 27, x FOR PEER REVIEW 7 of 38 using the methods reported in the literature [30,72,73]. Unfortunately, these methods did not produce the intended complexes reliably (i.e., poor yield, unwanted by-products, and product isolation difficulty); therefore, method development was required to obtain the most efficient method for isolating the complexes [71]. To prepare 1-8, the appropriate precursor, [Pt IV (HL)(AL)(OH)2] 2+ , was reacted with 2-3.5 mol eq. of the NHS esters in DMSO and left stirring for 72 h at room temperature in the dark ( Figure 8). Each reaction was washed with excess Et2O to afford two layers: a colourless and an oily brown layer. The colourless layer was discarded while the oily brown layer was collected and diluted with a minimum amount of MeOH, followed by the addition of excess Et2O to afford a beige precipitate. The precipitate was collected and washed with excess C3H6O to remove any unreacted or excess NHS esters and acids. Further purification using flash chromatography was required to obtain higher purity. Characterisation to confirm the successful synthesis  Table 1.   Table 2. A total of 10-11 peaks, including the solvent peak, were recorded for the studied complexes. No amine protons were observed due to proton exchange with D2O.  Table 1. A summary of experimental yields (%), HPLC peak areas (%), T R (min), and mass-to-charge ratios (m/z) of the studied complexes.  Table 2. A total of 10-11 peaks, including the solvent peak, were recorded for the studied complexes. No amine protons were observed due to proton exchange with D 2 O. Elucidating 1 as an example, the 1 H-NMR spectrum displays slightly shifted downfield multiplicity originating from the phen and DACH protons, which is attributed to the  Figure 9). The doublets at 9.22 and 9.17 ppm, with calculated J-coupling constants of 5.6 and 5.5 Hz, were assigned to aromatic protons H2 and H9, respectively. H4 and H7 resonated as a triplet at 9.03 ppm, while H3 and H8 protons appeared as a multiplet as shown in Figure 9. These resonances are typically observed as doublets for the platinum(II) and platinum(IV) precursors, PHENSS and PHENSS(IV)(OH) 2 , respectively [21]. We postulate that the differences in multiplicity are a consequence of axial coordination of the 4-CPA ligand, thereby shifting the chemical resonances more downfield due to a deshielding effect, and this was also evident in the 1 H-NMR spectra of 2 ( Figure S25).

Platinum(IV) Complexes
Molecules 2022, 27, x FOR PEER REVIEW 8 of 38 Elucidating 1 as an example, the 1 H-NMR spectrum displays slightly shifted downfield multiplicity originating from the phen and DACH protons, which is attributed to the coordination of 4-CPA to the platinum ( Figure 9). The doublets at 9.22 and 9.17 ppm, with calculated J-coupling constants of 5.6 and 5.5 Hz, were assigned to aromatic protons H2 and H9, respectively. H4 and H7 resonated as a triplet at 9.03 ppm, while H3 and H8 protons appeared as a multiplet as shown in Figure 9. These resonances are typically observed as doublets for the platinum(II) and platinum(IV) precursors, PHENSS and PHENSS(IV)(OH)2, respectively [21]. We postulate that the differences in multiplicity are a consequence of axial coordination of the 4-CPA ligand, thereby shifting the chemical resonances more downfield due to a deshielding effect, and this was also evident in the 1 H-NMR spectra of 2 ( Figure S25).  Two sharp doublets were also observed at 6.53 and 6.28 ppm, with the same calculated J-coupling constant of 8.3 Hz ( Figure 9). These sharp doublets were from the aromatic protons of 4-CPA, represented by a, b, c, and d. This indicates that the 4-CPA ligand was successfully coordinated to the intended scaffold. During the preliminary stages of assigning peaks, it was not clear where the peak for the methylene protons (e) of 4-CPA resonated. Although, by referring to the multiplet at 3.13 ppm in Figure 9 where DACH protons, H1 and H2 , resonated, a proton integration of 4 was recorded indicating that two extra protons were present. As a result, it was concluded that e overlapped with the peaks of H1 and H2 . To further confirm this, 2D-COSY was undertaken ( Figure S22). From the expanded 2D-COSY spectra of 1 (Figure 10), there is long-distance coupling between e and H1 and H2 at 3.13 ppm. This was also observed in the 2D-COSY of 2 ( Figure S26).
assigning peaks, it was not clear where the peak for the methylene protons (e) of 4-CPA resonated. Although, by referring to the multiplet at 3.13 ppm in Figure 9 where DACH protons, H1′ and H2′, resonated, a proton integration of 4 was recorded indicating that two extra protons were present. As a result, it was concluded that e overlapped with the peaks of H1′ and H2′. To further confirm this, 2D-COSY was undertaken ( Figure S22). From the expanded 2D-COSY spectra of 1 (Figure 10), there is long-distance coupling between e and H1′ and H2′ at 3.13 ppm. This was also observed in the 2D-COSY of 2 ( Figure  S26).     Figures S34-S38). 19 F-NMR was also carried out as an additional study for 3 and 4, to confirm further the presence of the F atom in the complexes (3: Figure S29; 4: Figure S34). Furthermore, the 1 H-NMR spectrum of the studied complexes (1-8) was stacked together, as presented in Figure 12, for comparison purposes. It is evident that the signals originating from the heterocyclic ligands (phen and 5,6-Me 2 phen) and DACH are consistent amongst the complexes. Of further note is the distance of signal splitting in the aromatic region (6-7 ppm), wherein the coordinated ligands resonated ( Figure 12). Evidently, the greater the size of the halogen that is on the PAA ligand and the less electronegative it is, the greater the splitting is observed, as highlighted in Figure 12.

UV and CD Measurements
The electronic transitions detected in the UV measurements are consistent with published examples [21,[25][26][27]71,74]. To attain consistency and accuracy, the UV spectra of complexes 1-8 ( Figures S55-S62) were obtained by titrations for each complex in triplicate. Molar extinction coefficients were also calculated based on a generated plot curve of absorbance against concentration using the Beer-Lambert law equation. Standard deviations were also calculated. In addition to UV absorption, CD measurements were also undertaken to confirm the retention of chirality of the ancillary ligand, SS-DACH. The obtained CD spectra ( Figures S63-S70) are also consistent with published examples [21,[25][26][27]71,74]. All characteristic peaks in the UV and CD spectra of the complexes are summarised in Table 3.

UV and CD Measurements
The electronic transitions detected in the UV measurements are consistent with published examples [21,[25][26][27]71,74]. To attain consistency and accuracy, the UV spectra of complexes 1-8 ( Figures S55-S62) were obtained by titrations for each complex in triplicate. Molar extinction coefficients were also calculated based on a generated plot curve of absorbance against concentration using the Beer-Lambert law equation. Standard deviations were also calculated. In addition to UV absorption, CD measurements were also undertaken to confirm the retention of chirality of the ancillary ligand, SS-DACH. The obtained CD spectra ( Figures S63-S70) are also consistent with published examples [21,[25][26][27]71,74]. All characteristic peaks in the UV and CD spectra of the complexes are summarised in Table 3. Table 3. Band maxima in the UV and CD spectra of 1-8.

Platinum(IV) Complexes
UV/λ max nm (ε/M·cm −1 ± SD × 10 4 ) CD/λ max nm (∆ε/M·cm −1 × 10 1 ) The influence of the halogens in the axial ligands of 1-8 produced slight differences or patterns based on their UV spectra ( Figures S55-S62). The results obtained from the UV measurements demonstrated both π-π* transitions and metal-to-ligand charge transfer interactions. The studied complexes are aromatic-rich because of the presence of heterocyclic ligands (phen and 5,6-Me 2 phen), which exhibit ligand-centred π-π* transitions. The UV spectra of platinum(IV) derivatives that contain phen, such as 1, 3, 5 and 7, exhibited similar absorption bands ( Figure 13). However, 3 demonstrated a strong absorption band at 204 nm, which was not observed for 1, 5, and 7, as shown in Figure 13. This band was also observed in their platinum(IV) precursor complex, PHENSS(IV)(OH) 2 [27]. Another observation was the slight bathochromic effect, or red shift exhibited at 245 nm for 7. These changes suggest that the phen derivatives containing F and I atoms had influenced slight observable shifts (3: strong band at 204 nm; 7: bathochromic shift at 245 nm) compared to the phen derivatives containing Cl and Br (1 and 5, respectively). Furthermore, the UV spectra of platinum(IV) derivatives that contain 5,6-Me 2 phen (2, 4, 6 and 8) also exhibited similar absorption bands, specifically the weak shoulder-like features between 220 and 250 nm followed by prominent bands between 280 and 290 nm ( Figure 14). A prominent peak was observed for 2 at 287 nm, which is broader compared to what 4, 6, and 8 exhibited at that wavelength ( Figure 14). Interestingly, the slight shifts between 220 and 250 nm shown in Figure 14 may be attributed to the halogens; however, these shifts were not demonstrated for the phen derivatives, 1, 3, 5, and 7. Of further note, at lower wavelengths (circa 207 nm), 2, 4, 6, and 8 did not produce the bands typically observed for their platinum(IV) precursor complex, 56MESS(IV)(OH) 2 [27]. Again, this confirms that the halogens slightly influenced the absorption bands recorded for the studied complexes.
peak was observed for 2 at 287 nm, which is broader compared to what 4, 6, and 8 exhibited at that wavelength ( Figure 14). Interestingly, the slight shifts between 220 and 250 nm shown in Figure 14 may be attributed to the halogens; however, these shifts were not demonstrated for the phen derivatives, 1, 3, 5, and 7. Of further note, at lower wavelengths (circa 207 nm), 2, 4, 6, and 8 did not produce the bands typically observed for their platinum(IV) precursor complex, 56MESS(IV)(OH)2 [27]. Again, this confirms that the halogens slightly influenced the absorption bands recorded for the studied complexes.   Chirality is crucial to the biological activity of the studied complexes. It has been previously confirmed that by replacing the chiral ancillary ligand SS-DACH with its enantiomer RR-DACH in the structure of our complexes, it significantly impacts on the cytotoxicity [17,18,20,75]. For example, in the murine leukaemia cell line (L1210), 56MERR and 56MESS elicited GI50 values of 460 and 9 nM, respectively, demonstrating a 51-fold difference in cytotoxicity [75]. In the same cell line, an almost 12-fold difference in cytotoxicity was demonstrated between PHENRR (1500 nM) and PHENSS (130 nM) [17].
CD measurements were undertaken in this study mainly to confirm that the SS-DACH of the studied complexes has been retained. Complexes 1-8 produced strong negative absorptions bands at lower wavelengths and weak positive bands at higher wavelengths ( Figures S63-S70). These results are consistent with published examples of platinum(IV) complexes, as they also exhibit negative absorption bands at lower wavelengths and positive absorption bands at higher wavelengths [21,[25][26][27]71,74]. By contrast, plati- Chirality is crucial to the biological activity of the studied complexes. It has been previously confirmed that by replacing the chiral ancillary ligand SS-DACH with its enantiomer RR-DACH in the structure of our complexes, it significantly impacts on the cytotoxicity [17,18,20,75]. For example, in the murine leukaemia cell line (L1210), 56MERR and 56MESS elicited GI 50 values of 460 and 9 nM, respectively, demonstrating a 51-fold difference in cytotoxicity [75]. In the same cell line, an almost 12-fold difference in cytotoxicity was demonstrated between PHENRR (1500 nM) and PHENSS (130 nM) [17].
CD measurements were undertaken in this study mainly to confirm that the SS-DACH of the studied complexes has been retained. Complexes 1-8 produced strong negative absorptions bands at lower wavelengths and weak positive bands at higher wavelengths ( Figures S63-S70). These results are consistent with published examples of platinum(IV) complexes, as they also exhibit negative absorption bands at lower wavelengths and positive absorption bands at higher wavelengths [21,[25][26][27]71,74]. By contrast, platinum(IV) complexes containing RR-DACH generally demonstrate positive absorption bands at lower wavelengths and negative absorption bands at higher wavelengths [27,74].

Solubility and Stability
In drug development, solubility and stability are important parameters that have a critical role in drug safety and effectiveness [76][77][78]. Since the complexes presented in this study, 1-8, incorporate axial ligands that contain different halogens (F, Cl, Br, and I), we investigated their solubility and stability in aqueous solution to see the impact of the halogens. Complexes 1-8 were found to be soluble in d.i.H 2 O at room temperature (Table 4). Interestingly, slight variations in their solubility were demonstrated, as shown in Table 4. Apart from 1, the derivatives containing F and Cl (2, 3, and 4) were more soluble than those derivatives containing Br and I (5, 6, 7, and 8) in d.i.H 2 O. Furthermore, the 5,6-Me 2 phen derivatives (2, 4, 6, and 8) were found to be more soluble than the phen derivatives (1, 3, 5, and 7) in d.i.H 2 O, which may also be directly influenced by the methylation of the heterocyclic ligand. While the methylation of the heterocyclic ligand and the halogenated axial ligands did alter the solubility of the complexes, their effects were not significant.  The stabilities of 1-8 were also evaluated using HPLC. The complexes were dissolved in 10 mM of PBS (~7.4 pH) and incubated at 37 • C to mimic physiological conditions. The process was followed for 36 h. Based on the HPLC chromatograms obtained ( Figure S79), no significant reduction to their platinum(II) and platinum(IV) precursor complexes was observed, confirming their stability in solution for 36 h. Overall, the halogenated axial ligands and the methylation of the heterocyclic ligand did not impact the stability of the complexes.

Lipophilicity
For lipophilicity measurements, HPLC was utilised instead of the traditional shakeflask method, as described previously [21,26,29]. Recent studies have also reported the efficiency of HPLC in measuring the lipophilicity of compounds [79,80]. Each complex was eluted under several isocratic ratios. A standard curve was generated to calculate the log value of the capacity factor (log k) according to Equation (1). Following Equation (2), the percentage of organic solvent (CH 3 CN) in the mobile phase from each isocratic run was plotted against log k w ( Figure S80). Log k w is the chromatographic lipophilicity index or measure of lipophilicity of the complex, so a higher value corresponds to greater lipophilicity [26,81,82]. Because the selected axial ligands in this study contain F, Cl, Br, and I, it was only appropriate to determine the influence of the halogens on the overall lipophilicity of the platinum(IV) complexes. According to the calculated log k w values (Table 5), the phen derivatives (1, 3, 5, and 7) demonstrated lower lipophilicity values compared to their 5,6-Me 2 phen counterparts (2, 4, 6, and 8) as expected, and this trend is consistent with the literature data [21,26,29]. This only indicates that the presence and absence of methyl groups in the heterocyclic ring system of the complexes play a role in altering lipophilicity. The most lipophilic complex was the 5,6-Me 2 phen derivative, 8, which contains 4-IPA, demonstrating a log k w of 1.19. Conversely, the least lipophilic complex was the phen derivative containing 4-FPA, 3, with a log k w of 0.25. Furthermore, for the phen derivatives, the order of increasing lipophilicity was 3 > 1 > 5 > 7, which corresponds to the order of increasing size of the halogens, F > Cl > Br > I. This pattern was also observed with the 5,6-Me 2 phen derivatives (4 > 2 > 6 > 8), thereby confirming that the halogens increased the order of lipophilicity accordingly based on their size.

Reduction Experiments
Platinum(IV) complexes are reduced to their platinum(II) precursors by intracellular biological reducing agents such as AsA or GSH in tumour cells [33,[83][84][85]. However, the precise mechanism by which reduction occurs is not fully understood. In this paper, reduction reaction experiments were undertaken on the synthesised platinum(IV) complexes, 1-8, with the aid of 1 H-NMR and 1D-195 Pt-NMR spectroscopy. The method used for the reduction reaction experiments was adapted from the literature without modifications [21]. A total of 10 mM PBS (~7.4 pH) was selected as media, and AsA was used as the biological reducing agent to investigate the reduction property of 1-8. Prior to the reduction reaction experiments, a control was prepared where each metal complex was dissolved with PBS in D 2 O. 1D-195 Pt-NMR was measured within the regions of −2800 and 400 ppm (30 min per region), at 37 • C. This was undertaken to confirm that the PBS would not interfere with the reduction process of the metal complexes. For the preliminary 1D-195 Pt-NMR spectra obtained ( Figures S81-S82), all complexes were unchanged with no sign of reduction, thus confirming that PBS does not reduce the complexes to their corresponding platinum(II) and platinum(IV) precursor complexes. The approximate time points for the studied complexes at which an estimated 50% reduction had occurred, in the presence of AsA, as represented by T 50% , are summarised in Table 6.
The reduction property of 1 was monitored for 1 h by 1 H-NMR spectroscopy at 37 • C ( Figure 15). Upon treatment of AsA, the complex started reducing at 5 min as shown in Figure 15. Between 15-20 min, approximately 50% reduction was demonstrated by 1 (Table 5). Accordingly, the resonances of the phen protons (H2 and H9; H4 and H7; H5 and H6; H3 and H8) and the aromatic protons of the 4-CPA ligand (a, b, c, and d) were almost equivalent (Figure 15). At 1 h, the metal complex had almost entirely reduced to its platinum(II) precursor, PHENSS. A notable observation from the 1 H-NMR experiments is the movement of resonances, particularly the evident upfield shift from the phen protons and the downfield shift from the 4-CPA protons, as shown in Figure 15. Table 6. A summary of approximate time points in min for 1-8 at which 50% reduction occurs as represented by T 50% .

Platinum(IV) Complexes
T 50% (min) 1 15-20 Subsequently, after the 1 H-NMR experiments, 1D-195 Pt-NMR was also carried out centred on the regions of −2800 and 400 ppm, at 37 °C. After 1 h from the final 1 H-NMR experiment, 1 had reduced entirely to PHENSS ( Figure 16). As shown in Figure 16, no platinum(IV) resonance was recorded in the 400 ppm region. Additionally, a platinum(II) resonance was recorded in the −2800 ppm region, also validating its complete reduction. The results suggest that 1 could be reduced inside tumour cells and that it could exert antitumor effects via its platinum(II) precursor, and is, therefore, a potential prodrug of PHENSS. Subsequently, after the 1 H-NMR experiments, 1D-195 Pt-NMR was also carried out centred on the regions of −2800 and 400 ppm, at 37 • C. After 1 h from the final 1 H-NMR experiment, 1 had reduced entirely to PHENSS ( Figure 16). As shown in Figure 16, no platinum(IV) resonance was recorded in the 400 ppm region. Additionally, a platinum(II) resonance was recorded in the −2800 ppm region, also validating its complete reduction. The results suggest that 1 could be reduced inside tumour cells and that it could exert antitumor effects via its platinum(II) precursor, and is, therefore, a potential prodrug of PHENSS.
Subsequently, after the H-NMR experiments, 1D-Pt-NMR was also carried out centred on the regions of −2800 and 400 ppm, at 37 °C. After 1 h from the final 1 H-NMR experiment, 1 had reduced entirely to PHENSS ( Figure 16). As shown in Figure 16, no platinum(IV) resonance was recorded in the 400 ppm region. Additionally, a platinum(II) resonance was recorded in the −2800 ppm region, also validating its complete reduction. The results suggest that 1 could be reduced inside tumour cells and that it could exert antitumor effects via its platinum(II) precursor, and is, therefore, a potential prodrug of PHENSS.  Furthermore, the reduction property of 2 was also investigated. The reduction kinetics of 2 was similar to 1 ( Figure 17). From the 1 H-NMR spectra shown in Figure 17, the complex began to reduce at 5 min, and new signals originating from its platinum(II) precursor and the aromatic protons of the axial ligand started to appear. About 50% reduction had occurred between 20 and 25 min for 2 (Table 6). After 1 h, 2 had almost reduced and the signals from the prodrug had decreased significantly while the signals from the released or uncoordinated 4-CPA acid became more prominent, further confirming that it had been detached from the platinum centre. Additionally, 1D-195 Pt-NMR was also carried out centred on the regions of −2800 and 400 ppm, at 37 • C ( Figure 18). While no platinum(IV) resonance was recorded at −400 ppm, a platinum(II) resonance was acquired at −2800 ppm, which confirms that 2 was able to entirely reduce back to its platinum(II) precursor, 56MESS.
Molecules 2022, 27, x FOR PEER REVIEW 18 of 38 Furthermore, the reduction property of 2 was also investigated. The reduction kinetics of 2 was similar to 1 ( Figure 17). From the 1 H-NMR spectra shown in Figure 17, the complex began to reduce at 5 min, and new signals originating from its platinum(II) precursor and the aromatic protons of the axial ligand started to appear. About 50% reduction had occurred between 20 and 25 min for 2 (Table 6). After 1 h, 2 had almost reduced and the signals from the prodrug had decreased significantly while the signals from the released or uncoordinated 4-CPA acid became more prominent, further confirming that it had been detached from the platinum centre. Additionally, 1D-195 Pt-NMR was also carried out centred on the regions of −2800 and 400 ppm, at 37 °C ( Figure 18). While no platinum(IV) resonance was recorded at −400 ppm, a platinum(II) resonance was acquired at −2800 ppm, which confirms that 2 was able to entirely reduce back to its platinum(II) precursor, 56MESS.   Figures S93 and S94). Interestingly, 6 and 7 reduced the slowest of the derivatives, wherein T50% occurred at 30 min (Table 6), and traces of platinum(IV) species were still observed in their 1D-195 Pt-NMR spectra even 1 h after the final 1 H-NMR experiments (Figures S90 and S92). By contrast, 3 and 8 reduced the fastest, wherein T50% was observed to occur between 5 and 10 min ( Table 6, and Figures  S83 and S93). The results confirm that the axial ligands' halogenation influenced the complexes' reduction behaviour more than methylation of the heterocyclic ligand. With the exception of 3 and 8, it can also be deduced that the derivatives with the most electronegative halogens in their axial ligands (Cl, F, and Br) reduced more quickly than the 4-IPA derivatives containing the least electronegative element, I. In summary, complexes 1-8 are thus potential prodrugs of their platinum(II) precursors, PHENSS and 56MESS.

Growth Inhibition Assays
Complexes 1-8 and the halogenated PAA ligands, 4-CPA, 4-FPA, 4-BPA, and 4-IPA, were screened for antiproliferative activity against a panel of cell lines that included HT29 colon, U87 glioblastoma, MCF-7 breast, A2780 ovarian, H460 lung, A431 skin, Du145 prostate, BE2-C neuroblastoma, SJ-G2 glioblastoma, MIA pancreas, the ADDP-resistant ovarian variant, and the non-tumour derived MCF10A breast line. Compound growth inhibition was assessed using the MTT assay after 72 h of treatment. A summary of the determined GI50 values is reported in Table 7, including the GI50 values of cisplatin, oxaliplatin, and carboplatin, as well as the platinum(II) and platinum(IV) precursors obtained from a previous study [21], for comparison purposes. It is already established that 56MESS is the most cytotoxic platinum(II) scaffold of its class, demonstrating an average GI50 value of 36 ± 10 nM from the panel of cell lines tested, as shown in Table 7. PHENSS was of lower potency than 56MESS, and this difference in cytotoxicity is attributed to be the effect of methylation in the 5 and 6 positions of the heterocyclic ligand [16,21,24,73]. Briefly, the structural differences between PHENSS and 56MESS are modest with changes in the pKa of the coordinated nitrogen (N) atoms in their heterocyclic ligands that could modulate  Figures S93 and S94). Interestingly, 6 and 7 reduced the slowest of the derivatives, wherein T 50% occurred at 30 min (Table 6), and traces of platinum(IV) species were still observed in their 1D-195 Pt-NMR spectra even 1 h after the final 1 H-NMR experiments (Figures S90 and S92). By contrast, 3 and 8 reduced the fastest, wherein T 50% was observed to occur between 5 and 10 min ( Table 6, and Figures S83 and S93). The results confirm that the axial ligands' halogenation influenced the complexes' reduction behaviour more than methylation of the heterocyclic ligand. With the exception of 3 and 8, it can also be deduced that the derivatives with the most electronegative halogens in their axial ligands (Cl, F, and Br) reduced more quickly than the 4-IPA derivatives containing the least electronegative element, I. In summary, complexes 1-8 are thus potential prodrugs of their platinum(II) precursors, PHENSS and 56MESS.  Table 7, including the GI 50 values of cisplatin, oxaliplatin, and carboplatin, as well as the platinum(II) and platinum(IV) precursors obtained from a previous study [21], for comparison purposes. It is already established that 56MESS is the most cytotoxic platinum(II) scaffold of its class, demonstrating an average GI 50 value of 36 ± 10 nM from the panel of cell lines tested, as shown in Table 7. PHENSS was of lower potency than 56MESS, and this difference in cytotoxicity is attributed to be the effect of methylation in the 5 and 6 positions of the heterocyclic ligand [16,21,24,73]. Briefly, the structural differences between PHENSS and 56MESS are modest with changes in the pKa of the coordinated nitrogen (N) atoms in their heterocyclic ligands that could modulate the overall positive 2 charge. Whilst at the molecular surface and conformational standpoint 5,6-methylation induces a negligible impact, as denoted by pKa values (i.e., phen = 4.9 versus 5,6-Me 2 phen = 5.6) [86], methylation significantly abrogates N-lone pair localization. As previously reported by Ramírez et al. [86] and others, pKa values are congruent with a variety of other parameters, which include π acceptor potential, charge accepting properties (µ+), and electro-accepting power (ω+) (high π acceptor character = high capacity to accept charge). Comparatively, demethylation confers a significant modulatory effect on these parameters according to the values extrapolated by Ramirez et al. on phen-based cobalt(II) coordination complexes [86].

Growth Inhibition Assays
Furthermore, the cancer-killing properties of 56MESS and PHENSS still surpass that of cisplatin, oxaliplatin, and carboplatin. For example, 56MESS and PHENSS were 1100-fold and 70-fold more reactive, respectively, compared to cisplatin in the cisplatin-resistant HT29 colon cancer cell line (Table 7). In the ADDP-resistant ovarian variant cell line, 56MESS and PHENSS proved to be 2150-fold and almost 150-fold more potent than cisplatin, respectively. Notably, 56MESS exhibited a GI 50 value of 4.6 ± 0.4 nM in the Du145 prostate cell line, demonstrating almost 3300-fold and 630-fold increases in potency over carboplatin (15,000 ± 1200 nM) and oxaliplatin (2900 ± 400 nM), respectively.
The platinum(IV) precursor complexes, PHENSS(IV)(OH) 2 and 56MESS(IV)(OH) 2 , still demonstrate potency, but not to the same degree as their platinum(II) precursors.  (Table 7). Table 7. A summary of the GI 50 values (nM) of 1-8, 4-CPA, 4-FPA, 4-BPA, and 4-IPA in multiple cell lines. The data presented for cisplatin, oxaliplatin, carboplatin, and precursor platinum(II) and platinum(IV) precursors were obtained from a previous study [21]. nd = not determined. For the new series of platinum(IV) complexes incorporating the halogenated PAA ligands, the most cytotoxic derivatives contained the methylated heterocyclic ring system, 5,6-Me 2 phen (2, 4, 6 and 8), whereas the least cytotoxic derivatives contained the unmethylated heterocyclic ring system, phen (1, 3, 5, and 7), as expected. This observation supports the notion that methylation of the heterocyclic ligand is a critical contributor to tuning the overall biological activity of the complexes. For the axial ligands, 4-CPA, 4-FPA, 4-BPA, and 4-IPA, their cytotoxicity was also tested; however, they did not demonstrate significant cytotoxicity (Table 7). Amongst the platinum(IV) derivatives synthesised, 4, which contains the 4-FPA ligand, and 6, which contains the 4-BPA ligand, exhibited the greatest cancerkilling potential with GI 50 values ranging from 0.7 to 110 nM in the panel of cell lines tested ( Table 7). It is also noteworthy that the difference in reactivity between 4 and 6 is marginal, especially in the A2780 ovarian, ADDP-resistant ovarian variant, and H460 lung cancer cell lines, where both displayed the same GI 50 values. Additionally, 4 and 6 displayed their lowest GI 50 values in Du145 prostate, MIA pancreas, HT29 colon, and the ADDP-resistant ovarian variant cancer cell lines, as shown in Table 7. Particularly, in the Du145 prostate cancer cell line, 4 exhibited an GI 50 value 0.7 ± 0.4 nM, which is 6.6-fold more potent than its platinum(II) precursor, 56MESS (4.6 ± 0.4 nM), and about 1700-fold more potent than cisplatin (1200 ± 100 nM). In the same cell line, 6 (1 ± 0.6 nM) also proved to be 4.6 times more potent than 56MESS and 1200 times more potent than cisplatin. This suggests that 4 and 6 may be most effective in treating prostate cancer. From the results, it can also be inferred that the conjugation of 4-FPA and 4-BPA to 56MESS(IV)(OH) 2 enhances the overall activity of the complexes. Furthermore, the third-most cytotoxic platinum(IV) derivative in the group contained the 4-CPA ligand (2). 4-CPA has been previously investigated as a safe and effective treatment against estrogen-sensitive breast cancer [63][64][65]. It was our expectation that when 4-CPA is coordinated to 56MESS(IV)(OH) 2 , a prodrug that demonstrates potent cytotoxicity response towards the estrogenic MCF-7 breast cancer cell line can be generated. Complex 2 displayed a GI 50 value of 180 ± 77 nM in that cell line, which is almost 2-fold less potent than its platinum(II) precursor, 56MESS (93 ± 44 nM) ( Table 7). The potency of 56MESS in all of the cell lines remains unsurpassed compared to 2. Nevertheless, 2 showed selectivity towards the HT29 colon, MIA pancreas, and ADDP-resistant ovarian cancer cell lines instead of the MCF-7 breast cancer cell line. This indicates that the coordination of 4-CPA to 56MESS(IV)(OH) 2 did not significantly influence overall cytotoxicity unlike 4-FPA and 4-BPA.
The remaining platinum(IV) derivatives (1, 3, 5, 7, and 8) may not be as reactive as 2, 4, 6, or 56MESS, but they are still significantly more potent than cisplatin, oxaliplatin, and carboplatin, and the degree of difference is substantial (Table 7). Intriguingly, the 5,6-Me 2 phen derivative, 8, which contains 4-IPA, had an average GI 50 value of 387 ± 50 nM over the cell lines tested, sharing almost the same cytotoxicity values with the phen derivatives 1, 3, 5, and 7. Evidently, the coordination of 4-IPA to 56MESS(IV)(OH) 2 did not enhance the overall activity of the complex. Furthermore, the phen derivatives, 1, 3, 5, and 7 were as potent as their platinum(II) precursor, PHENSS, which suggests that the presence of the axial ligands had little to no impact on the overall activity of the complexes. By contrast, an overall improvement of activity was demonstrated when 4-CPA, 4-FPA, and 4-BPA were coordinated to 56MESS(IV)(OH) 2 , generating the most cytotoxic platinum(IV) derivatives, 2, 4, and 6. Moreover, 1-8 are potent in the ADDP-resistant ovarian variant cell line in comparison to all other cell line populations, indicating that the complexes are not susceptible to the drug resistance mechanisms induced by standard clinical treatment with cisplatin.

ROS Production
Although ROS is required for cancer cell function and progression, it can induce programmed cell death or apoptosis [87]. Modulating the levels of ROS may treat cancer by causing DNA damage, resulting in mitochondrial membrane permeabilization that subsequently signals to the execution caspases (3, 6, and 7) to cleave the DNA repair protein PARP-1 (116 kDa), which produces two fragments (89 and 24 kDa) that reduce the binding affinity and repairing abilities [88,89]. In this study, we report the ROS activity of the studied platinum(IV) complexes, 1-8, together with the individual axial ligands (4-CPA, 4-FPA, 4-BPA, and 4-IPA) in the human cancer cell line, HT29 colon ( Figure 19, Table 8, and Figure S95). The ROS potential of their platinum(II) and platinum(IV) precursors in the same cell line is also presented for comparison purposes (Table 8) [21]. The HT29 colon cell line was selected as it is one of the human cancer cell lines where complexes 1-8 demonstrated superb antitumor potential (Table 7). HT29 colon cells were stained by DCFH-DA and then treated with the complexes at each specific GI 50 concentration. Upon ROS production, the DCFH-DA produced dichlorodihydrofluorescein, a fluorescent product. The measured fluorescence was, therefore, proportional to the produced ROS. Table 8. ROS production upon treatment with 1-8 in HT29 colon cells at 24, 48, and 72 h. The data presented for platinum(II) (PHENSS and 56MESS), platinum(IV) (PHENSS(IV)(OH) 2 , and 56MESS(IV)(OH) 2 ) precursors were obtained from a previous study [21].

Materials and Methods
All chemicals and reagents were of analytical grade and used without further purification. The deionised water (d.i.H2O) used for the experiments was obtained from a Mil-liQ TM system (Millipore Australia Pty Ltd., Sydney, NSW, Australia).  Complexes 1-8 showed significant production of ROS at 24, 48, and 72 h (p < 0.0001), with 4 and 6 producing the greatest ROS of 738 ± 15 RFU and 643 ± 14 RFU at 72 h, respectively ( Figure 19, Table 8, and Figure S95). The exceptional surge of ROS produced by 4 and 6 may be causative of cytotoxicity potency, as they elicited GI 50 values of 6 ± 2 and 7 ± 2 nM, respectively, in the HT29 cancer cell line (Table 7). In comparison, HT29 cells treated with the ligands 4-CPA, 4-FPA, 4-BPA, and 4-IPA exhibited an increase in ROS production relative to the control at 24, 48, and 72 h ( Figure 19, Table 8, and Figure S95).
The results confirm that the studied platinum(IV) complexes, 1-8, may potentially induce DNA damage through the production of ROS inside cancer cells and subsequently induce apoptosis.

Materials and Methods
All chemicals and reagents were of analytical grade and used without further purification. The deionised water (d.i. Retention times (T R ) were measured at varying isocratic ratios ranging from 30 to 36% of solvent B at 1 mL·min −1 mL. An injection volume of 10 µL was utilised. Capacity factors were determined according to Equation (1): where k is the capacity factor, T R is the retention time of the analyte, and T 0 represents the dead time. A minimum of four different mobile compositions were used for each complex to calculate k. A linear plot was generated of log k against the concentration of CH 3 CN in the mobile phase to determine the value of log k w expressed by Equation (2): where S is the slope, ϕ is the concentration of the CH 3 CN in the mobile phase, and log k w represents the capacity factor of the complex in 100% d.  1D-195 Pt-NMR was measured using a spectral width of 85,470 Hz and 674 data points. 19 F-NMR was acquired at eight scans with a spectral width of 75,188 Hz and 300,742 data points. All recorded resonances were presented as chemical shifts in parts per million (δ ppm) with J-coupling constants reported in Hz. For spin multiplicity: s (singlet); d (doublet); t (triplet); q (quartet); and m (multiplet). All spectroscopic data gathered were generated and plotted using TopSpin 4.1.3 analysis software.
The reduction properties of 1-8 were monitored using 1 H-NMR and 1D-195 Pt-NMR spectroscopy on a 400 MHz Bruker (Melbourne, VIC, Australia) Avance Spectrometer. A sequence of 1 H-NMR experiments was carried out for 1 h at 37 • C, followed by 1D-195 Pt-NMR within the regions of −2800 and 400 ppm. An amount of 10 mM PBS (~7.4 pH) was transferred to a vial and reduced to dryness through rotary evaporation. AsA (~1 mg) was combined with the metal complex (~5 mg) and transferred to the vial containing the dried PBS. A total of 600 µL of D 2 O was then added to the vial to dissolve the complex, AsA, and the PBS together. Each reaction was followed at 37 • C until the complete reduction of the complexes.

Ultraviolet-Visible (UV) Spectroscopy
An Agilent (Melbourne, VIC, Australia) Technologies Cary 3500 UV-Vis Multicell Peltier spectrophotometer was utilised to perform the UV spectroscopy experiments. UV spectroscopic experiments were completed at room temperature in the range of 200-400 nm with a 1 cm quartz cuvette. All complexes were prepared in d.i.H 2 O. A stock solution of each complex (1 mM) was prepared and absorption spectra were recorded at a series of different concentrations by titrating 9 × 3 µL aliquots into a cuvette containing d.i.H 2 O (3000 µL). Experiments were repeated in triplicate. All spectra were baseline-corrected by the instrument-a baseline containing d.i.H 2 O was acquired first, and automatically subtracted from each experiment. Average extinction coefficients (ε) were determined with standard deviation and errors based on the generated plot curves.

Circular Dichroism (CD) Spectroscopy
A Jasco (Easton, PA, USA) J-810 CD spectropolarimeter was used to measure the CD spectra of the studied prodrugs. The samples were prepared in d.i.H 2 O in a 1 mm optical glass cuvette or 1 cm quartz cuvette. CD experiments were undertaken at room temperature in the wavelength range of 200-400 nm (30 accumulations) with a bandwidth of 1 nm, data pitch of 0.5 nm, a response time of 1 sec and a 100 nm·min −1 scan speed. The flowrate of nitrogen gas was 6 L·min −1 . The HT (photo-multiplier) level remained below 500 V for all experiments. A CD simulation tool (CDToolX) was used to generate the spectra. All NHS esters were synthesised as previously described [70,71], but with minor modifications. The acids were reacted with 1 mol eq. of both NHS and DCC in either C 3 H 6 O or DCM. The reaction solution was left stirring in a covered flask at room temperature for 24 h. The precipitated by-product, dicyclohexylurea (DCU), was removed either through vacuum filtration or syringe filtration. The filtrate was collected and rotary evaporated to dryness. All NHS esters were used without further purification.

ROS Detection Assay
To investigate the presence of ROS in treated cells, a DCFDA/H2DCFDA-cellular ROS Assay Kit (Abcam, Cambridge, MA, USA) was used, as previously described [21,[91][92][93]. A total of 25,000 cells/mL of HT29 cells in DMEM were seeded in 96-well plates. Cells were washed with 1X kit buffer, and then stained with 25 µM 2 ,7 -dichlorofluorescein diacetate (DCFH-DA) and incubated for 45 min. DCFH-DA was then removed, and cells were then re-washed with 1X kit buffer, after which phenol red free media was added. Cells were then treated with a GI 50 drug concentration for each complex. The plates were directly scanned to measure fluorescence (relative fluorescence units (RFU)) at different time points using the Glo-Max ® -Multimode microplate reader (Promega Corporation, Alexandra, VIC, Australia) at an excitation/emission of 485/535 nm. To generate the positive control (20 µM tert-butyl hydroperoxide (TBHP)), cells were washed with 1X kit buffer, and stained with DCFDA (25 µM) for 45 min; this was removed and TBHP was added in phenol red free media, and the resulting solution was scanned as described above.

Conclusions
The structure and purity of eight novel platinum(IV) complexes (1-8) incorporating halogenated PAA derivatives, 4-CPA, 4-FPA, 4-BPA, and 4-IPA, were confirmed by HPLC, NMR, UV, CD, and ESI-MS. NMR was used to monitor the reduction behaviour of the complexes in the presence of AsA. HPLC provided insights into the stability and lipophilicity of the complexes. All complexes proved to be soluble in aqueous solution, and surprisingly stable for 36 h at 37 • C in 10 mM PBS (~7.4 pH). As for the lipophilicity results, the phen derivatives were less lipophilic than the 5,6-Me 2 phen derivatives. The order of increasing lipophilicity of the complexes also parallels the order of increasing size of the halogens incorporated into the axial ligands (F < Cl < Br < I). No correlation was demonstrated between lipophilicity and cytotoxicity of the complexes. The in vitro results also confirmed that 1-8 surpassed the potency of cisplatin and its derivatives in all of the cell lines tested.
Notably, 4 and 6 demonstrated strong selectivity towards the cell lines HT29 colon, Du145 prostrate, MIA pancreas, ADDP-resistant ovarian, and MCF10A, with GI 50 values ranging between 0.7 and 11 nM. In the ADDP-resistant ovarian variant cell line, 4 and 6 were almost 4700-fold more potent than cisplatin. There was some correlation between ROS potential and cytotoxicity in the HT29 colon cell line. The most biologically active complex, 4, demonstrated the most significant production of ROS, while the least cytotoxic complex, 3, demonstrated the lowest observable production of ROS. In summary, the findings of this study are a testament that modifying certain structural features is essential in tuning the biological activity of novel therapeutic molecules. Furthermore, stability and reduction experiments on the studied complexes in human blood plasma or serum using NMR are scheduled in the near future.

Patents
This work is part of Australian Provisional Patent Application 2022900110, Platinum(IV) complexes, February 2022, Western Sydney University, Sydney, Australia.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/molecules27207120/s1, Figure S1: HPLC chromatogram of NHS-4-CPA within the collection wavelength of 254 nm obtained at 298 K, by an Agilent ZORBAX RX-C 18 column (100 × 4.6 mm, 3.5 µm pore size) using a 0-100% linear gradient over 15 min with a flow rate of 1 mL·min −1 . Figure S2: HPLC chromatogram of NHS-4-FPA within the collection wavelength of 254 nm obtained at 298 K, by an Agilent ZORBAX RX-C 18 column (100 × 4.6 mm, 3.5 µm pore size) using a 0-100% linear gradient over 15 min with a flow rate of 1 mL·min −1 . Figure S3: HPLC chromatogram of NHS-4-BPA within the collection wavelength of 254 nm obtained at 298 K, by an Agilent ZORBAX RX-C 18 column (100 × 4.6 mm, 3.5 µm pore size) using a 0-100% linear gradient over 15 min with a flow aromatic protons of the 4-FPA ligand as indicated by the red arrows. T represents time in min. Figure S84: 1D-195 Pt-NMR spectra of 3 with 10 mM PBS (~7.4 pH) and AsA in D 2 O, within the regions of -2800 and 400 ppm at 310.15 K, highlighting its complete reduction after 1 h from the final 1 H-NMR experiment. Figure S85: 1 H-NMR spectra of 4 with 10 mM PBS (~7.4 pH) and AsA in D 2 O at 310.15 K, in different time intervals, highlighting the movement of resonances from the phenanthroline protons and the aromatic protons of the 4-FPA ligand as indicated by the red arrows. T represents time in min. Figure S86: 1D-195 Pt-NMR spectra of 4 with 10 mM PBS (~7.4 pH) and AsA in D 2 O, within the regions of -2800 and 400 ppm at 310.15 K, highlighting its partial reduction after 1 h from the final 1 H-NMR experiment as indicated by *. Figure S87: 1